托佩克猪SLA-3原核表达载体构建及表达

Construction and Expression of the Prokaryotic Expressing Vector of SLA- 3 Derived from Topigs Pig

目的:研究托佩克猪SLA-3-TPK的原核表达载体的构建及蛋白表达。方法:设计引物扩增SLA-3-TPK胞外区(命名为SLA-3-TPKe),并将此片段克隆至p MD19-T Simple Vector,经双酶切筛选阳性克隆测序,序列正确的克隆片段与表达载体p ET-21a(+)连接,转化宿主菌BL21,并经过诱导表达,SDS-PAGE检测目的蛋白的表达。结果:PCR成功扩增获得SLA-3-TPke,大小约为850bp,酶切后插入片段大小约为830bp,经克隆测序,阳性克隆序列与原序列一致,双酶切鉴定证实目的基因与p ET-21a(+)成功连接,经过IPTG诱导表达和SDS-PAGE检测,结果显示目的基因成功表达且目的蛋白大小约为31k Da。结论:该研究成功构建了托佩克猪SLA-3原核表达载体,获得了表达蛋白,为今后进一步的结构和功能研究奠定基础。

Objective： To construct and express the prokaryotic expressing vector of SLA- 3- TPK. Method： A pair of primers was designed to amplify the extracellular domain of SLA- 3- TPK（ named SLA- 3- TPKe）,and then the amplified product was cloned into p MD19- T Simple Vector. After cleaved by NdeⅠ and XhoⅠ,the positive clones were selected to be sequenced. Analyzing by biological soft,the cloned product with correct sequence was selected to inserted into p ET- 21a（ ＋） and transformed into BL21. After induction and expression,the interest of protein was detected by SDS- PAGE. Result： The PCR result was shown that the SLA- 3- TPKe was amplified successfully and the molecular weight was about 850 bp and the inserted fragment was about 830 bp. After sequencing and analysis,the sequence of the positive clone of SLA- 3- TPKe was consistent with the primary sequence. By cleavage,the interest of gene was proved to be successfully inserted into the p ET- 21a（ ＋）. After induction with IPTG and SDS- PAGE detection,the interest of gene was expressed and the molecular weight was about 31 k Da. Conclusion： This research has constructed the prokaryotic expressing vector of SLA- 3 derived from To Pigs pigs and the interest of protein was obtained,which will lay a base to study the structure and function of the SLA- 3- TPK in future.